Enantiomers

 Enantiomers are non superimposable mirror

images.
 Molecules are non superimposable if there
is no orientation in which all atoms of both
molecules can be superimposed
Examples of enantiomers
Properties of Enantiomers:

 In general, enantiomers have the same

physical properties (B.p, m.p, density, etc).
 Enantiomers will rotate plane polarized light
the same magnitude () but in opposite
directions (+ or -).
 Enantiomers can have significantly different
biological properties
 Ibuprofen

R form(non-active) side effects S form(active) anti inflammatory

Thalidomide is now being used to treat plasma cell cancer,
leprosy, and has shown anti-HIV activity
RACEMIC MIXTURE
 A racemic mixture is a 50:50 mixture of two
enantiomers. As they are mirror images, each
enantiomer rotates plane-polarized light in
an equal but opposite direction.
 A sample with only a single enantiomer is an
enantiomerically pure, enantiopure or
homochiral compound.Racemic mixtures can
be symbolized by a (d/l)- or (+/-)- prefix in
front of the substance's name
RACEMIC MIXTURE SINGLE ENANTIOMER
Amlodipine Levamlodipine
Amphetamine Dextroamphetamine
Bupivacaine Levobupivacaine
Cetirizine Levocetirizine
Citalopram Escitalopram
Fenfluramine Dexfenfluramine
Formeterol Arformeterol
Ibuprofen Dexibuprofen
Ketamine Esketamine
Ketoprofen Dexketoprofen
Methyl phenidate Dexmethylphenidate
Milnacipran Levomilnacipran
Modafinil Armodafanil
Ofloxacin Levofloxacin
Omeprazole Esomeprazole
Zopiclone Eszopiclone
chlorpheniramine dexchlorpheniramine
Need to separate
enantiomers
 Preparation of enantiopure (ee~100%)
compounds is one of the most important aims
both for industrial practice and research.
 As enantiomers of drug substances may have
distinct biological interactions and,
consequently, profoundly different
pharmacological, pharmacokinetic, or
toxicological activities.
 The body is highly chiral selective; it will
interact with each racemic drug differently
and metabolize each enantiomer by a
separate pathway to produce different
pharmacological activity.
Need to separate
enantiomers

 One isomer may thus produce the desired

therapeutic activities, while the other may be
inactive or produce unwanted side effects.
 Even when side effects are not serious, the
inactive enantiomer must be metabolized and
thus represents an unnecessary burden for the
organism.
Methods of seperation

 Resolution of racemic compounds

 Crystallization
 Chiral chromatography
 Capillary electrophoresis
 Asymmetric synthesis/Enantioselective
synthesis
 Enzymes
 Kinetic resolution
 Polymeric membranes
 1.Resolution
 Seperation of racemic mixture in to individual enantiopure
compounds is called as “resolution”.
 Since enantiomers have common physical properties( B.p,
m.p,solubility) they cannot be seperated by methods like
crystallization, distillation or basic chromatography.
 Diastereomers have different physical properties ,so they
can be seperated easily.
 This fact is used to achieve resolution of racemates
 Reaction of a racemate with an enantiomerically pure chiral
reagent gives a mixture of diastereomers, which can be
separated
Resolution

Four general strategies that take advantage

of formation of diastereomeric
interactions to separate racemic mixture:

1. Formation of diastereomeric salts with an

enantiopure resolving agent
2. Formation of diastereomeric compounds
with an enantiopure resolving agent
3. Use of chiral stationary phases
4. Enzymatic resolution
Diastereomeric salt formation

 Louis Pasteur was able to isolate the stereoisomers of

tartaric acid because they crystallize from solution as
crystals with differing symmetry and shape.
 (S)-1-Phenylethylamine combines with a racemic
mixture of tartaric acid to form diastereomeric salts
,which are seperated by crystallization.
 After seperation each of the diastereomers are treated
with strong acid(Example: HCL) to regenerate the
corresponding enantiomer of tartaric acid.
Diastereomeric salt formation
Formation of diastereomeric
compounds
 Racemic mixture + resolving
agentdiastereomeric compounds
 After seperation the resolving agent is recovered
by cleaving the covalent bond formed in the
earlier step.
 Menthol is used as a resolving agent in the
formation of diastereomeric esters of mandelic
acid
Chiral stationary phases
 Another method to resolve racemic mixture
through chromatography on chiral stationary
phases.
 These are incorporated in GC and liquid
chromatography.
 In the resolution of 2-amino butane on
chromatographic system in which an
enantiomer of mandelic acid is attached to a
stationary phase ,new and transient
diastereomeric interactions between 2-amino
butane and the stationary phase lead to
different retention times and thus to
seperation of enantiomers
 Enzymatic resolution
 Resolution of racemic mixtures by the action of enzymes.
 For example, lipase acts on a kilogram of racemic starting
material to generate enantio-enriched products that may
be seperated by distillation after filtration of the enzyme
from the reaction mixture.
 2.Asymmetric autocatalysis

 Through selective autocatalysis ,enantiomers can also

be seperated.
 example: the soai reaction that achieves a form of
synthetic seperation of enantiomers through selective
autocatalysis.
 In this reaction, the racemate treated with a catalyst
and after several cycles ,it gives almost enantiopure
sample.
Soai reaction
 3.Chiral pool synthesis
 Chiral pool synthesis is one of the simplest and oldest
approaches for enantioselective synthesis.
 Chiral pool synthesis is a strategy that aims to improve the
efficiency of enantioselective synthesis. It starts the
organic synthesis of a complex enantiopure chemical
compound from a stock of readily available enantiopure
substances
 A readily available chiral starting material is manipulated
through successive reactions, often using achiral reagents, to
obtain the desired target molecule.
 This can meet the criteria for enantioselective synthesis when
a new chiralspecies is created.
Chiral pool synthesis
4.Kinetic resolution

 Simple method
 Racemate is subjected to a reaction using chiral
agent and one of the enantiomers of the racemate
reacts more quickly than the other.
 example: An enantioenriched alcohol is obtained
from a racemate through the addition of an
enantiopure acylating agent
 identical physical
properties

Pd-catalyzed Oxidation

easy to separate
5.Crystallization

 The racemic compound is reacted with a resolving

agent
 So, the mixture contains three chiral compounds :
two enantiomeric compounds and another chiral
compound.
 At this time the more stable diastereomer
crystallizes and it can be separated from the non
racemic enantiomeric mixture by several methods.
Resolution of racemic
menthol
1.Crystallization without any solvent:
• The mixture of resolving agent(RA) and racemic
compound should be warmed until it melted
• Then it should be cooled while one of the
diastereoisomers crystallizes from the melt.
• RA : O,O’-dibenzoyl-(R,R)-tartaric acid ((R,R)-
DBTA).
• The crystalline molecular complex
(diastereoisomer) contains the (1R,2S,5R)-menthol
(the Lmenthol
• While the remained melt is enriched in the other
enantiomer
2. Crystallization with solvent

 RA : (R,R)-DBTA.
 Solvent : Hexane
 The crystalline molecular complex
(diastereoisomer) contains the (1R,2S,5R)-
menthol (the L menthol).
 While the other enantiomer reamined in the
solvent.
 6.Capillary electrophoresis
 CE is a complementary technique to HPLC, particularly for
the analysis of charged and polar compounds.
 Enantiomer separations of volatile and thermostable chiral
compounds, and additional compounds that could be
derivatized without racemization could be performed
 Advantages of CE for chiral analyses include high efficiency,
simple instrumentation, low sample and reagent
consumption, and speed in method development and
analysis.
 For electrically driven chiral separations the selector can
be added in different ways.Fused silica capillaries are
routinely used in CE.
 Cyclodextrins are most frequently used. For crown ethers
,only small molecules bearing an amino group such as an
amino acids can be seperated
Capillary electrophoresis
• Application of high voltage (5 kV – 30 kV) across a
narrow bore (50 –75 mm i.d.) bare fused silica
capillary filled with conductive aqueous-based
buffer.
• Separation is achieved by overall differences in
analyte charge/mass ratios, giving rise to different
analyte velocities
• Chiral selectors (e.g., cyclodextrins (CDs) can be
added directly to the run buffer, leading to the
formation of transient diastereomeric complexes
with analytes
• Chiral separation is achieved by either differences
in the selector affinity between enantiomers or
differences in mobility of the diastereomeric
complexes.
• Development of chiral separation methods often
involves optimization of selector type, selector
concentration, different mixtures of selectors,
buffer pH, or buffer concentration
1.Low pH, negative polarity: Sulphated CDs have mobility to detector,
interact with positively charged and neutral compounds, imparting mobility
to detector.

2.Low pH, positive polarity: Neutral CD moves with EOF. Neutral CDs
interact with positively charged compounds, slowing their mobility
towards detector.
• 96-capillary array CE instrument with fixed wavelength
UV detection
• Unattended analysis of two 96-well sample plates
• Robotic interfacing capabilities
• For chiral separations, additional capillary cooling was
supplied by ducting cold air (water chilled to 4° C)
across the capillary array
Advantages of Multiplexed CE-UV for Drug
Discovery
•Simultaneous monitoring of up to 96 individual CE
separations

•Low UV wavelength (214 nm) provides more universal

analyte detection

•Multiple applications (e.g., pKa, log P, purity, chiral

screening, drug analysis) can be performed with minimal
changeover time

•Requires only small quantities of sample and buffer

additives

•Tolerant to sample impurities (CE is separation technique)

•Variation of buffer conditions (e.g., pH, ionic strength,

buffer additives, additive concentrations) in different
capillaries can significantly accelerate methods
development
• Multiplexed CE-UV is an attractive approach
for performing high throughput chiral
selector screening or chiral separations
• Up to 96 different selector/analyte
combinations can be evaluated in a single CE
experiment, significantly speeding method
development
• Good migration time and peak area
reproducibility can be achieved between
different capillaries of the array
• Low levels of enantiomeric impurities (<
0.5%) can be detected
• Optimized methods can be performed in
parallel or transferred to single capillary
instruments
Comparision between HPLC and CE
HPLC
Instrument, cost, and Expensive columns;
safety consumption of more buffers
and hazardous organic
substances
Chiral selectors Great amount of commercially
available CSP’s required.
Efficiency Efficiency sometimes poor
Seperation scale Semi preparative and preparative Analytical scale, very small
scale.
Reproducibility Good; high success rate.
Method development Relatively slow; changing and
conditioning a column is time-
consuming.
CE
Simple; no
pump,injector,valves;requir
es less solvent; low amount
chiral
selector;inexpensive;enviro
nmental friendly
Common and Inexpensive
chiral selectors
High peak efficiency
sample volumes
Relatively poor and low
success rate.
Rapid; changing a capillary
and=or chiral selector takes
only few minutes
 7.Seperation by HPLC
 Chromatographic methods have dominated separation of
enantiomers during the past several decades, especially HPLC
 Chiral HPLC is more versatile than chiral GC for enantiomeric
separation because it can separate a wide variety of non-volatile
compounds. It can be used to develop fast and accurate methods of
chiral drug separation, and it allows on-line detection and
quantitation of both mass and optical rotation of enantiomers when
appropriate detection devices are used
 Direct chiral separation using CSPs is more widely used and
predictable in mechanistic terms than methods involving chiral
additives in the mobile phase.
 To date, more than a hundred HPLC CSPs are commercially
available.
 No single CSP can be considered universal; none has the ability to
separate all classes of racemic compounds.
 HPLC
 HPLC remains the dominant technique for chiral separation
in industry
 HPLC is useful because of its preparative-scale capability.
 The method is generally slow and laborintensive, however,
requiring specialized engineering approaches for acceptable
throughout
 Three components should be considered in developing a
chiral separation method: analyte, CSP, and mobile phase.
 Chiral HPLC has been used to separate chlorpheniramine and
its main monodesmethyl metabolite, verapamil and its
metabolite, and tramadol and its metabolite
 HPLC

 HPLC is the most widely used technique for the separation

of enantiomers due to:
 Their extensive applications
 Ease of applicability
 Availability of advanced instrumentation
 Drawbacks:
 The use of large amounts of toxic solvents
 Potentially long equilibration and analysis times
 Significant peak broadening sometimes occurs as a result of
the relatively slow diffusion
8.Chiral seperations using GC

 Because of the high sensitivity, efficiency and speed,

chiral separation by capillary GC represents a versatile
method for enantiomer analysis.
 Chiral separations in GC can be achieved via:
 Using a derivatization procedure with a chiral auxiliary
 Using mobile phase additive
 to form diastereomer complexes which then separated
on an achiral stationary phase.
 Using a non-racemic chiral stationary phase (CSP)
 In the late 1960s, chiral separation in GC was a challenging
problem among scientists.
 In 1967, Prof. E. Gil-Av was successfully demonstrated chiral
separation for L- and D-amino acid esters with lauryl ester of
N- trifluoroacetyl-L-isoleucine as CSP.
 In 1977, the first polysiloxane bonded CSP, Chirasil-VAL
(thermal stability > 200⁰C) was developed by H. Frank, G.
Nicholson and E. Bayer.
 In 1987, the first capillary column coated with cyclodextrins
was invented by Prof. W. A. Konig in cooperation with
Macherey-Nagel.
 In the early of 1980s, the successful separations of o-, m-, p-
xylenes and ethylbenzene on a cyclodextrins CSP were
published.
 GC
 Factors affecting the efficiency of the column, the
resolution of peaks and the elution temperature are
important to consider when working with enantiomer
separation using GC
 The efficiency, usually expressed as theoretical plate
height,H, and resolution, Rs, provides a quantitative
measure of the ability to separate two peaks. H and Rs can
be calculated according to:
 H=L/N where L=length of the capillary and
N=no. of theoretical plates
 Because the resolution of peaks is dependent on the
efficiency of the column, factors affecting the efficiency
are also affecting resolution
 Based on their unique chiral selectors, many of
CSP in GC are classified into three main groups:
 chiral separation on non-racemic chiral
amino acid derivatives via hydrogen bonding
 chiral separation on non-racemic chiral
metal coordination compounds via
complexation
 chiral separation on biogenic cyclodextrin
derivatives via inclusion
Subsequently, all of the chiral selectors are
chemically linked to polysiloxanes
 Non-racemic amino acid derivatives as CSP
 Hydrogen bonding interaction
 Enantiomeric separation of polar analytes such as alcohols,
diketones, amines and 2- and 3-hydroxyl carboxylic acids.
 Peak switching principle
For example: on Chiral-L-Val, the D-AAs elute before the L-
AAs.
 Used for trace analysis and purity studies of amino acids
(AAs).
 Theenantiomer with a significantly lower concentration
must be eluted first for the quantification to be accurate.
 Upper temperature limit of 200⁰ C
 Drawback: racemization of the stationary phase.
derivatization of analytes in order to increase volatility and
interaction sites
 Chiral metal coordination compounds as CSP
 Metal coordination principles
 Using various chiral 1,3-diketonate bis
chelates of Mn(II), Co(II) and Ni(II)
 Chiralanalysis of volatile non-
hydrogen-bonding compounds
 Chiral analysis of chiral oxygen-,
nitrogen- and sulfur-containing
compounds.
 Used for mechanistic investigations and
for understanding inherent principles of
chiral recognition (entropy/enthalpy
compensation)
 Not widely used
 Cyclodextrins (CDs) as CSP
 Cyclic molecules consisting of six (α- CD), seven (β- CD) or
eight (γ-CD) D- glucopyranose units bonded through α- 1,4
linkages.
 Formed as enzymatic degradation products of starch.
 CDs can interact with an analytes via:
 Functionalized CDs
Hydrogen bonds (functional groups)
 Dipole/dipole interactions (functional groups)
 Hydrophobic interactions (carbon content)
 Steric interactions
 Unfunctionalized CDs
 Inclusion (size of the molecule)
Cyclodextrins (CDs) as CSP

Unfunctionalized CDs:
Lower possibility of formation of the opposite chirality
Lower chance of reversal elution

Drawback: Low solubilites from the

hydroxyl groups at positions 2, 3, and 6.
 Mixed Stationary Phases
 Using favorable interactions of each class
 Widening the range of analytes
 Often consist of two different selector classes, primarily
amino acid derivatives and inclusion-type structures, such as
CDs.
 Efficiencies of separation may decrease if the separations are
dependent on one type of interaction.
 Advantages and disadvantages
using GC
 Advantages
 No sample derivatization is required (except for very polar
analytes)
 High efficiency at high speed, sensitive
 Temperature-programming tools
 The CSPs need not be enantiomerically pure
 In contrast with liquid chromatographic and electrophoretic
methods, the optimization of mobile phases with respect to
solvents, pH of buffers, modifiers and gradients is absent.
 Development time and optimization is faster than for HPLC.
 Lower LODs,no solvent wastage
 Disadvantages:
- Volatility,
- Thermal stability
- Stereochemical integrity (not prone to racemization)
- Derivatization is required, if compound is polar
- Optically pure chiral reagent (ideally 99% optically pure)
 GC
 Chiral separations using GC represent a popular and advanced
technique.
 Chiral separations using GC employ two methods: (1) direct
approach utilizing chiral stationary phases; (2) indirect approach
requiring derivatization with a chiral reagent.
 The selection of a CSP still remains a matter of trial and error (no
universal CSP for GC is yet available).
 Permethylated β- cyclodextrin-derived CSPs appear to be leading
 New types of stationary phases are constantly being developed.
 The wide range of applications demonstrates that scientists from
widely differing disciplines find the technology useful.
 9.Polymeric membranes

 Liquid membranes with immobilized chiral ligand have also

been used for chiral separation although these techniques
could be difficult to apply in commercial systems because of
the instability of the liquid membranes
 An alternative approach is to use an affinity ultrafiltration
system in which a large stereoselective ligand is added to
the bulk solution to selectively bind, and thus retain, one of
the stereoisomers
 The mechanism of chiral separation on polymeric
membranes can be categorized as diffusion-selective
membranes and sorption-selective membranes
 Membranes
 Diffusion-selective membranes are usually made of an
intrinsically chiral polymer without specific foreign chiral
selectors, for example albumin or other proteins, chiral
polysaccharide chains or segments, DNA, crown ether
derivatives, and oligopeptides.
 Sorption-selective membranes can be made by
embedding or immobilizing chiral selectors in polymer
membranes or on the membrane surfaces and these
membranes have less selective diffusion but show highly
selective sorption. Examples of chiral selectors include
crown ether derivatives,cyclodextrin, albumin and other
proteins, and DNA
 Membranes
 Most studies of chiral separation membranes have been
performed in dialysis membranes.The main disadvantages of
the dialysis method are that the concentration of the final
product is more dilute than that of the feed solution, and that
permeation is extremely slow.
 Due to these disadvantages, chiral separation in industrial
applications may require ultrafiltration or nanofiltration
through chiral separation membranes.
 In addition to dialysis and filtration, pervaporation via
membranes is also useful for the chiral separation process,
where the driving force of the permeation is a vapor pressure
difference.
 Membranes
 Enantioselective vapor permeation is also effective for chiral
separation if the racemic compounds are more or less
volatile
 Several chiral separation membranes were prepared from
chiral polymers
 Chiral separation using membranes with immobilized large
molecules as chiral selectors can work by three mechanisms:
(1) affinity membranes,
(2) selective sorption membranes, and
(3) selective diffusion membranes
 Membranes
 Polypeptide membranes have shown very high enantiomer
permeation rates with encouraging selectivity for chiral
drug separation
 Immobilized cyclodextrins are also used. Compared with
other chiral selector-immobilized membranes, CD-
functionalized membranes have a lower cost and might have
wider applicability and higher tolerance in various
environments.
 However, chiral separation through immobilized CD
membranes has the disadvantage of low selectivity because
native cyclodextrins have limited chiral recognition ability
and limited flexibility, which are important to enable
interaction with the enantiomers
Molecularly imprinted
polymers(MIP’s)
 Advantages
 Robust with high mechanical strength
 Resistant to elevated pressure or temperature
 Stable in the presence of extreme acids,bases or organic
solvents
 Have special recognition sites with predetermined
selectivity for the analyte
 Drawbacks
 Low chromatographic efficiency
 High peak asymmetry
 10.Super critical fluid
chromatography
 SFC has been used successfully for chiral
seperations at the analytical,semipreparative and
preparative scales.
 Commercial systems have demonstrated excellent
performance, robustness and cost effectiveness.
 For industrial purposes,SFC at a simulated moving
bed(SMB) on production scale has been
demonstrated on a prototype in the lab
 The production capacity can be obtained at metric
tons level.
Characteristics of super critical
fluids(SFC’s)
 They are considered as green mobile phases because of
their :
 Limited environmental impact
 Reduced consumption of toxic solvents and additives
 Lack of toxicity
 Cost reduction of solvents and waste removal
 Residue free removal of the solvent from the extract
 Advantages of SFC over HPLC
 High speed
 Higher through put
 Used with wide range of sensitive detectors
 Higher column efficiency
 Smaller volumes of toxic solvents and hazardous waste
 Improved resolution
 Extended temperature capability
 Higher flow rates
 Can be used for analytical and preparative scales
 Smaller pressure drop across the column
Disadvantages of SFC
 The temperature and pressure must be carefully
controlled
 Expensive technology
 Recent technique that still requires further
developments
 SFC is suitable for non-polar pharmaceuticals and
cannot be applied to polar compounds
 This can be corrected by addition of an organic
modifier to the mobile phase e.g.
• Methanol
• Acetonitrile
• Methylene chloride
Types of columns used for SFC

 Three types of columns used :

 Capillary columns
 Open tubular columns
 Packed columns
 Multiple chiral stationary phases(CSP’s) and
combinations of both chiral and achiral stationary
phases can be coupled in series in SFC to achieve the
desired selectivity.
Mobile phase:Cabondioxide is mostly used as it has low
viscous nature,environmental friendly,and high diffusivity.
Types of chiral selectors used for
SFC
 They can be classified in to :
1.Macromolecular
 Derivatized polysaccharides
 Proteins
 Synthetic polymers
2.Macrocyclic
 Cyclodextrins
 Macrocyclic antibiotics
3.Low molecular weight
 Pirkle type CSP’s
Poly saccharide based chiral
selectors
 Of the many polysaccharide based chiral selectors ,
Amylose and cellulose are recommended due to their
abundance and excellent capabilities for chiral resolution.
 Amylose provides more chiral grooves for enantiomeric
resolution
 These are used for determination of various enantiomer-
enantiomer interactions and also for developing and
designing homochiral drugs.
 Chiralpak AD; Chiralpak AS; Chiralcel OD;Chiralcel OJ are
the polysaccharide chiral selectors mostly used because of
their enantioselectivity,high loading capacity and their use
in preparative scale
 cyclodextrins

 These are homochiral,non-ionic,cyclic oligosaccharides

 Composed of 6-12 D-(+)-glucopyranose units connected through
alpha -1,4- linkages.
 Alpha ,beta and gamma cyclodextrins are most commonly used
containing 6,7 and 8 glucopyranose units.
 Cyclodextrin chiral stationary phases causes efficient
separation.
 example: using cyclodextrins Aminoglutethimide separation is
done by SFC;where as thalidomide by HPLC.
Glycopeptide-based macrocyclic
antibiotics

 Examples: vancomycin,teicoplanin,ristocetin
Advantages
 They contain several stereogenic centres and functional
groups
 Their basket like geometry allows inclusion type
complexation
 They are amphoteric
 They are hydrophobic and hydrophilic in nature so can
be used in NP and RP modes.
Applications of SFC

 Powerful analytical and preparative technique for chiral

seperations and purifications e.g. caffeine and
ketoconazole
 Quantifying minor pollutants in chiral drugs
 Can be used in the quality control of chiral
pharmaceuticals
 In-vivo analysis of chiral drugs and their metabolites e.g.
warfarin,ketoprofen,propranolol
 In forensic science,SFC is helpful for identification of
synthesis or isolation sources of illicit materials e.g.
methamphetamine
 SFC over HPLC

 It has been demonstrated that SFC is rapidly replacing

HPLC in many pharmaceutical situations not only for
purification but also as the standard screening and method
development tool for chiral compounds for the following
reasons:
 Often provides faster seperations than HPLC
 Easily transposed to the preparative scale
 Provides high flow rate due to low viscosity
 Cost, health and safety benefits due to reduction in the
use of organic solvents.
 CONCLUSION
 It is necessary to consider the chiral nature of a compound in drug
research and development and the drug regulatory process.
Enantiomers of all chiral bioactive molecules must be separated and
tested.
 Chiral techniques have been developed for the separation and analysis
of chiral drugs. Among these, HPLC based on CSPs is widely employed
for the assays of drug enantiomers in pharmaceutical preparations and
biological fluids. More recently, CE plus chiral selector additives to the
running buffer have been used.
 In both chromatographic and electrophoretic methods, different types
of chiral selectors, including CDs, crown ethers, quinine, chiral
surfactants, polysaccharides, proteins, and macrocyclic antibiotics,
have successfully been used for chiral separation of drugs and drug
candidates.